Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco‐engineering and monoclonal antibody production

Abstract

SummaryBiopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core a(1,3)-fucose and b(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two a(1,3)-fucosyltransferase (FucT) and the two b(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked b(1,2)-xylose and had a significant reduction in core a(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core a(1,3)-fucose and b(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

“…At a later time, the publication by Li et al . (), showing that knocking out FucT 1 + 2 by TALENs does not completely abolish FucT activity, brought the genes FucT 3, 4 and 5 to our attention. FucT 3, 4 and 5 cluster together and have low nucleotide sequence identity with FucT 1 and 2, but the genes share the conserved catalytic fucosyltransferase motif in exon 4 (Jost et al ., ).…”

“…With the advent of designer nucleases such as zinc finger nucleases (ZFNs) (Kim et al ., ), transcription activator‐like effector nucleases (TALENs) (Christian et al ., ), and most recently CRISPR‐related nucleases like Cas9 (Jinek et al ., ) and Cpf1 (Zetsche et al ., ), it has become increasingly straightforward to knock out the functions of multiple genes. In N. benthamiana , the two XylT genes and two of the five FucT genes were knocked out with TALENs to completely eliminate the β‐1,2‐xylosyltransferase activity and reduce core α‐1,3‐fucosyltransferase activity by 60%, the latter confirming that the other FucT genes would need to be targeted to eliminate FucT activity completely (Li et al ., ). Two recent publications describe the successful multiplex CRISPR‐mediated knockout of four or five FucT genes and two XylT genes in Nicotiana tabacum BY‐2 suspension cells (Hanania et al ., ; Mercx et al ., ), resulting in the absence of α‐1,3‐fucose and β‐1,2‐xylose in endogenous and recombinant proteins, and no differences in cell morphology or growth between the knockout and wild‐type lines.…”

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

“…TALEN-or CRISPR/Cas9-mediated gene knockouts have been made in diverse plant species amenable to transformation (Brooks et al, 2014;Fauser et al, 2014;Forner et al, 2015;Gao et al, 2015;Christian et al, 2013;Liang et al, 2014;Shan et al, 2013;Sugano et al, 2014;Xu et al, 2015;Zhang et al, 2014). Traits of commercial value have also been created by targeted knockout of selected genes Clasen et al, 2016;Haun et al, 2014;Li et al, 2012Li et al, , 2016. Alternatively, DSBs can be repaired by homologydependent repair (HDR), which copies information from a "donor" DNA template.…”

New traits in crops produced by genome editing techniques based on deletions

“…CRISPR-Cas9 is particularly suitable to multiplexing because of the versatility of the gRNAs, but also TALENs have been multiplexed: four Nicotiana benthamiana genes involved in glycosylation were knocked out simultaneously using TALENs for the benefit of biopharmaceutical production of glycoproteins devoid of plant-specific residues (Li et al 2016). Peterson et al (2016) targeted 14 loci simultaneously in A. thaliana with CRISPR-Cas using stacked gRNA expression arrays (Peterson et al 2016).…”